pollen studies in the genus viola (violaceae) from iran

Acta Bot. Croat. 73 (1), 93–106, 2014 CODEN: ABCRA25ISSN 0365-0588

eISSN 1847-8476

Pollen studies in the genus Viola (Violaceae) from Iran

SHAHRYAR SAEIDI MEHRVARZ1*, NARJES YOUSEFI1, MARYAM MOHAMMADI1,THOMAS MARCUSSEN2

1 Department of Biology, Faculty of Science, University of Guilan, P. O. Box41335-1914, Rasht, Iran.

2 Department of Plant and Environmental Sciences, University of Gothenburg, P. O. Box461, SE 405 30 Gothenburg, Sweden.

Abstract – Pollen morphology of 17 species of Viola representing five sections, Mela-

nium, Plagiostigma, 'Spathulidium' ined., Sclerosium, and Viola, was studied using lightand scanning electron microscope. Pollen grains were usually symmetrical, tetrazonocol-porate to pentazonocolporate in section Melanium and trizonocolporate to tetrazonocol-porate in the other four sections. Pollen shape was circular to subtriangular, tetragonal orpentagonal in polar view and prolate to oblate, spheroidal or pyramidal in equatorial view.Exine ornamentation was granulate, psilate and mostly perforate. The psilate type was onlyobserved in V. modesta. We found heteromorphy in aperture number in V. caspia of sectionViola, V. occulta of section Melanium and V. behboudiana of section Sclerosium, whichcorroborates their higher ploidy than in related species (octoploid versus tetraploid).

Key words: Exine, morphology, ornamentation, palynology, pollen, Viola

Introduction

Viola L. is the largest genus of Violaceae, with ca 600 species and is the only one widelydistributed in the Northern Hemisphere (BALLARD et al. 1999). Infrageneric classificationhas varied, but recent phylogenetic analysis indicates that the genus can be subdivided intotwo subgenera and 16 sections worldwide (YOUSEFI et al. 2012). Five sections belonging tosubgenus Viola are present in Iran: section Melanium Ging., section Plagiostigma Godr.,section Viola, section Sclerosium W. Becker, and the undescribed section 'Spathulidium'

ined. The three first sections are widespread in the Northern Hemisphere and together com-prise a few hundred species, while the two last sections are restricted to parts of southwest-ern Asia with fewer than a dozen species. The sections are ancient allotetraploid except forsection 'Spathulidium' which is inferred to be allooctoploid (MARCUSSEN et al. 2011). Re-cently, molecular phylogenetic analyses of sect. Melanium (including 25 species) were per-formed using ITS sequences and ISSR markers (YOCKTENG et al. 2003); they have shown

ACTA BOT. CROAT. 73 (1), 2014 93

* Corresponding author, e-mail: [email protected]® 2014 by Acta Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved.

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that sect. Melanium forms a derived and monophyletic group. Section Melanium, the pan-sies, is represented in Iran by five annual-ephemeral species, V. arvensis, V. kitaibeliana, V.modesta, V. occulta, and V. tricolor. The only species of section Plagiostigma occurring inIran, V. somchetica, belongs to subsect. Patellares (Boiss.) Rouy and Foucaud. Section Vi-

ola is restricted to northernmost Iran, where it occurs with six species, V. alba (subsp. alba),V. odorata, and V. sintenisii in subsect. Viola and V. caspia, V. reichenbachiana, and V.rupestris in subsect. Rostratae. Section Sclerosium is distributed in the South of Iran withthree putative species, V. behboudiana, V. cinerea and V. stocksii. Section 'Spathulidium’

ined. refers to a small group of three central Asian species, in Iran represented by V.

spathulata and V. pachyrrhiza; V. maymanica occurs in northern Afghanistan (SCHMIDT

1992). 17 species of Viola are distributed in different parts of Iran, mainly in the north. Ofthese, only V. spathulata is endemic to the territory (Elbrus mountains), but another fivespecies are endemic to the region.

The delimitation of Viola species within sections and subsections can be problematic inmany regions of the world, owing partly to phenotypic plasticity, partly to hybridisation andpolyploidy, and partly to the few morphological characters separating taxa. Introgressionappears, however, to be rare and restricted to certain groups (VALENTINE 1962, KRAHUL-

KOVÁ et al. 1996, MARCUSSEN et al. 2001, HILDEBRANDT et al. 2006).Pollen morphology of Violaceae in general has been examined by some authors

(ERDTMAN 1952, PETTET 1964, WALKER and DOYLE 1975). A few additional studies havebeen published on pollen morphology in Viola. GORB (1994) studied 12 Ukrainian speciesof Viola sect. Melanium (including V. arvensis, V. kitaibeliana, and V. tricolor) using bothlight and scanning electron microscopy. PERVEEN and QAISER (2009) examined five Viola

species (including V. stocksii) from Pakistan. DINÇ (2009) studied two species of subsectionViola (V. sandrasea, V. kizildaghensis) endemic to Turkey. Pollen heteromorphism is de-fined as the production by the same plant of pollen grains differing in aperture number(TILL-BOTTRAUD et al. 1995). This phenomenon occurs in over 30% of angiosperm species(MIGNOT 1994). In Viola pollen heteromorphism occurs in about a third of the studied spe-cies and appears to be associated with polyploidy (NADOT et al. 2000).

The main aim of this study is to provide a descriptive investigation of pollen grains in thegenus Viola and also of pollen heteromorphism in the species distributed in Iran, using lightand scanning electron microscopy. In light of the nomenclatural confusion and paucity ofmorphological characters for discrimination of Viola taxa in Iran (YOUSEFI et al. 2012),pollen morphology and heteromorphism may provide new characters useful for the delimi-tation of these taxa. In this study, pollen morphology of 13 species of the genus Viola withthe exception of V. arvensis, V. kitaibeliana, V. tricolor and V. stocksii are described for thefirst time.

Material and methods

Pollen of 24 taxa representing the 17 species of Viola occurring in Iran was studied bymeans of light microscopy (LM) and scanning electron microscopy (SEM). Pollen sampleswere obtained from herbarium specimens collected the same year. In order to ensure theconstancy of pollen characters among different populations of a certain species, 2–3 speci-mens per population were included in the analysis; otherwise each species was representedby only one population. The voucher specimens were deposited in Guilan University Her-

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MEHRVARZ S. S., YOUSEFI N., MOHAMMADI M., MARCUSSEN T.



barium. A list of the voucher specimens is given in table 1. Pollen grains were stained withbasic-fuchsine, then mounted in glycerin jelly and prepared for LM observation. Some mea-surements i.e. polar axis (P), equatorial axis (E), colpus length and exine thickness weremade by LM (Nikon microscope model Optiphot-Z optic) for 30 pollen grains under a mag-nification of 1000x. For analysing pollen heteromorphism, the number of apertures werecounted for 100 pollen grains, then the percentage of each aperture-class –three, four andfive– were calculated and the percentage lower than 95 was reported as heteromorphic pol-len (MIGNOT 1994).

For SEM observations, pollen grains soaked in absolute ethanol were pipetted directlyon to 12.5 mm diameter stubs and air-dried at room temperature; they were then coated in asputter coater with approximately 25 nm of Gold-Palladium. The micrographs were ob-

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POLLEN STUDIES IN THE GENUS VIOLA (VIOLACEAE) FROM IRAN

Tab. 1. Collection data of Viola species examined in the present. Species endemic to Iran are indi-cated by an asterisk (*).

Species Sect. / Subsect. Collection data

V. arvensis

MurrayMelanium Ging. Iran: Mazandaran, Javaherdeh, Jirkoh, May 1999,

Naderifar, GUH-13682V. kitaibeliana

Schult.Iran: Mazandaran, East slope of Damavand, above

Malar, June 2000, Moosavi & Mozafarian, TARI-33184V. modesta

FenzlIran: Kohgiluye-Boyer Ahmad, Nourabad to Yasuj road,

30 km to Yasuj, May 2009, Yousefi, GUH-13263-1V. occulta Lehm. Iran: Guilan, Deylaman, May 2009, Yousefi & Saeidi,

GUH-13683V. tricolor L. Iran: Guilan, Rudbar, April 2009, Ghahremani,

GUH-13663V. somchetica

C. KochPlagiostigma Godr./Estolonosae Kupffer

Iran: Ardebil, Almas road, April 2009, Yousefi & Shahi,GUH-13674

V. behboudiana

Rech. F. et Esfand.Sclerosium Iran: Baluchestan, Hudar, Ghasre ghand, 380 m,

Foroughi, TARI-10755V. cinerae

Boiss.Iran: Bushehr, 2–3 km of NE Khormoj, 150 m,

Mozafarian, TARI- 17141V. stocksii

Boiss.Iran: Hormozgan, Bandar-Abas, 20–30 km of W Rudan,

200 m,Wendelbo & Foroughi, TARI-15630V. spathulata

Willd.*«Spathulidium« ined. Iran: Mazandaran, Chalous-Karaj road, Duzdbon, April

1977, Ghahreman & Aghoostin, TUH-11252V. pachyrrhiza

Boiss. et Hohen.Iran: Lorestan, Aligudarz road to Shoolabad, 2400 m,

Runemark & Lazari, TARI-26228V. caspia (Rupr.)

Freyn 1Viola/Rostratae

(Kupffer) W. BeckerIran, Guilan,, Lahijan, Sheitan koh, March 2008,

Tabarestani, GUH-13634V. caspia (Rupr.)

Freyn 2Iran: Guilan, Masouleh, Yousefi, GUH-4369

V. caspia (Rupr.)Freyn 3

Iran: Guilan, Lahijan, Sheitan koh, March 2007, Yousefi,GUH-13681

V. reichenbachiana

Jord. ex Bor.Iran, Guilan, Asalem to Khalkhal road, March 2009,

Yousefi & Asaadi, GUH-13651



tained from a LEO1430 VP (England) at an accelerating voltage of 10–15 kV under magni-fications of 2040× to 11190×. Pollen terminology according to WALKER and DOYLE (1975),PUNT (2007) and HALBRITTER et al. (2008) has been followed.

Results

General pollen characters of the genus Viola

The pollen grains were shed in monads, symmetrical, tetrazonocolporate to pentazono-colporate in section Melanium and trizonocolporate to tetrazonocolporate in the other foursections (Figs. 1–2, Tab. 2). The apertures were colporate, equatorially elongated andreached the poles of the pollen with rounded or acute ends.

The smallest pollen grains belonged to V. spathulata (P = 29 mm, E = 26.08 mm) and thelargest ones to V. arvensis (P = 69.09 mm, E = 68.23 mm). The P/E ratio varied between 0.94and 1.18 (Tab. 2). The exine thickness varied between 0.6 µm (V. spathulata) and 1 µm (V.

occulta).

Exine ornamentation proved to be a valuable taxonomic character in Viola. It variesfrom perforate, granulate, to psilate (Tab. 2). Among these patterns the simple psilate typerepresented by V. modesta is very characteristic.

Section Melanium (Figs. 1A–G, 3A–G)

Section Melanium is a morphologically well-defined group of about 125 species. Ofthese, five species are distributed in Iran, mostly in the north. Members of this section have

96 ACTA BOT. CROAT. 73 (1), 2014


Species Sect. / Subsect. Collection data

V. rupestris F. W.Schmidt

Iran: Ardebil, Almas road, April 2009, Yousefi &Asaadi, GUH-13672

V. alba Bess.subsp. alba 1

Viola/Viola Iran: Guilan, Masouleh, March 2008, Royan,GUH-13266


Iran: Guilan, Tootkabun, April 2009, Yousefi & Saeidi,GUH-13684


Iran: Guilan, Lahijan, Sheitan koh, 2008, Yousefi, GUH-13271

V. odorata L. 1 Iran: Guilan, mountains of Masouleh, March 2008,Yousefi & Saeidi, GUH-13665

V. odorata L. 2 Iran: Guilan, Masouleh road, March 2007, Yousefi &Saeidi, GUH-13481-2

V. sintenisii W.Becker 1

Iran: Guilan, Lahijan, Sheitan koh, February 2008,Yousefi, GUH-13675

V. sintenisii W.Becker2

Iran: Guilan, Lakan, February 2008, Yousefi,GUH-13281

V. sintenisii W.Becker 3

Iran: Guilan, Lashtenesha, the rice field, GUH-13296-2

Tab. 1. – continued



ACTA BOT. CROAT. 73 (1), 2014 97


Fig. 1. Micrographs of pollen grains in Viola arvensis (A–B), V. kitaibeliana (C), V. modesta (D), V.

occulta (E), V. tricolor (F–G), V. somchetica (H), V. behboudiana (I–J), V. cinerea (K), V.

stocksii (L). Scale bar: 10 µm.

Fig. 2. Micrographs of pollen grains in Viola pachyrrhiza (A), V. spathulata (B), V. caspia (C), V.

reichenbachiana (D–E), V. rupestris (F), V. alba (G), V. odorata (H), V. sintenisii (I). Scalebar: 10 µm.



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Tab. 2. Characteristic features of the investigated pollen in species of Viola. Abbreviations : P– polar axis (µm); E– equatorial axis (µm); M– mean value;SD – standard deviation; Eo – exine ornamentation; Pe: perforate, Gr: granulate, Ps: psilate.

Species Sect. / Subsect.E (mm)

Min (M ± SD) MaxP (mm)

Min (M ± SD) MaxP Eo

V. arvensis Melanium 60.0 (68.2 ± 5.1) 85.0 62.5 (69.1 ± 3.7) 77.5 1/01 Pe-GrV. kitaibeliana 42.5 (45.6 ± 1.8) 47.5 30.0 (43.2 ± 7.9) 55.0 0/94 –V. modesta 27.5 (34.8 ± 4.8) 40.0 40.0 (41.1 ± 3.2) 47.5 1/18 PsV. occulta 52.5 (60.0 ± 4.7) 67.5 55.0 (63.3 ± 4.0) 70.0 1/05 Pe-sub PsV. tricolor 50.0 (52.6 ± 2.1) 57.5 45.0 (54.9 ± 3.6) 62.5 1/04 GrV. somchetica Plagiostigma 25.0 (29.0 ± 1.5) 32.5 27.5 (31.9 ± 2.0) 35.0 1/1 Pe- sub PsV. behboudiana Sclerosium 29.4 (30.8 ± 1.1) 32.5 25.9 (29.5 ± 1.6) 31.7 0/95 Pe (fine)V. cinerae 32.5 (33.9 ± 1.0) 35.7 33.0 (38.2 ± 2.0) 40.0 1/14 Pe (fine and large)V. stocksii 38.2 (41.1 ± 1.7) 42.0 42.5 (44.0 ± 1.2) 45.0 1/07 Pe (fine)V. pachyrrhiza Spathulidium 23.0 (26.1 ± 1.7) 31.0 26.0 (29.0 ± 1.9) 34.0 1/11 Pe- LargeV. spathulata 22.5 (26.1 ± 1.7) 30.0 25.0 (29.0 ± 1.9) 32.5 1/11 GrV. caspia 1 Viola.Rostratae 32.5 (36.4 ± 1.7) 37.5 32.5 (36.5 ± 1.8) 40.0 1/00 (micro) GrV. caspia 2 27.5 (31.3 ± 2.2) 37.5 27.5 (33.5 ± 2.9) 37.5 1/07 GrV. caspia 3 40.0 (34.4 ± 3.6) 30.0 40.0 (35.5 ± 2.9) 32.5 1/03 GrV. reichenbachiana 27.5 (32.8 ± 3.7) 37.5 27.5 (32.6 ± 3.5) 37.5 0/9 Pe- sub GrV. rupestris 22.5 (25.3 ± 1.5) 30.0 27.5 (29.1 ± 1.6) 32.5 1/14 (micro) GrV. alba subsp. alba 1 Viola.Viola 25.0 (28.1 ± 1.9) 32.5 30.0 (31.6 ± 1.4) 35.0 1/12 Pe-sub PsV. alba subsp. alba 2 25.0 (30.3 ± 2.6) 35.0 27.5 (32.3 ± 3.0) 37.5 1/06 Pe- GrV. alba subsp. alba 3 25.0 (24.5 ± 1.1) 27.5 25.0 (28.8 ± 3.6) 30.0 1/17 Pe-sub PsV. odorata 1 25.0 (27.9 ± 2.6) 35.0 25.0 (29.5 ± 2.6) 35.0 1/05 GrV. odorata 2 25.0 (26.7 ± 1.3) 30.0 25.0 (31.6 ± 1.7) 35.0 1/18 (micro) Pe-GrV. sintenisii 1 25.0 (26.4 ± 2.0) 32.5 25.0 (30.3 ± 2.4) 35.0 1/14 Pe-GrV. sintenisii 2 22.5 (26.9 ± 3.7) 32.5 25.0 (30.6 ± 4.1) 37.5 1/13 Pe- GrV. sintenisii 3 25.0 (29.4 ± 3.7) 35.0 30.0 (33.8 ± 3.7) 40.0 1/14 Pe- Gr

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four to five apertures, while a more frequent aperture number in members of other sectionsis three. The pollen type of V. arvensis is perforate–granulate (Figs. 3A–B).

At species level, V. kitaibeliana with pyramidal shape in equatorial view (Fig. 1C) can bedistinguished from the other species, which are prolate to spheroidal in shape.

Exine ornamentation does not provide valuable characters in delimitation of sectionMelanium from other sections, but it is useful for species recognition within this section. Forinstance, two morphologically close species, V. tricolor and V. modesta, have different typesof exine sculpturing, i.e. granulate and psilate, respectively (Figs. 3C, F–G). Furthermore, V.

arvensis, which exhibits a perforate to granulate exine, can be separated from its relative V.

occulta, which has perforate to subpsilate exine (Figs. 3D–E).

Section Plagiostigma (Figs. 1H, 3H–I)

Viola somchetica is the only Iranian species of this section and its pollen grain in equato-rial view and exine ornamentation is prolate–spherodial (Fig. 1H) and perforate–subpsilate(Figs. 3H–I), respectively. From a palynological point of view, this species does not showany obvious morphological apomorphies.

Section Sclerosium (Figs. 1I–L, 3J–L, 4A–C)

Viola behboudiana is easily separated from the other Iranian species of the section bytwo palynological characters: the shape of pollen grains, which is oblate–spheroidal in V.

behboudiana (Figs. 1I–J) and prolate–spheroidal in V. cinerea and V. stocksii (Figs. 1K–L),

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Fig. 3. SEM micrographs of pollen grains in Viola arvensis (A–B), V. modesta (C), V. occulta (D–E),V. tricolor (F–G), V. somchetica (H–I), V. behboudiana (J–K), V. cinerea (L).



and the presence of pollen heteromorphism in V. behboudiana. All of the three species haveperforate to perforate–subpsilate exine ornamentation (Figs. 3J–L, 4A–C).

Section 'Spathulidium' ined. (Figs. 2A–B, 4D–F)

The two Iranian species of Section Spathulidium are morphologically similar but can beeasily separated using palynological characters: V. pachyrrhiza has perforate exine (Fig.4D), whilst V. spathulata has granulate exine ornamentation (Figs. 4E–F). Pollen mor-phology shows a basic similarity between section Spathulidium and section Plagiostigma;

however these two sections can be differentiated according to exine ornamentation (Tab. 2).

Section Viola

No palynological characters separate this section from the others.

Subsection Rostratae (Figs. 2C–F, 4G–L, 5A)

This subsection has three species in Iran, V. caspia, V. reichenbachiana and V. rupestris.The morphological recognition of the first two species is very problematic due to thevariable morphological characters; however palynological studies provide useful charactersfor delimiting these taxa. Pollen grains of V. caspia are prolate–spheroidal to subprolate inequatorial view (Fig. 2C) with a granulate exine (Figs. 4G–J), while V. reichenbachiana hasoblate–spheroidal pollen grains (Figs. 2D–E) with a perforate–subgranulate exine (Fig.4K). Furthermore, V. caspia shows pollen heteromorphism, in which the dominant pollenshape has three apertures and 6 % of pollen grains have four apertures. The characteristics ofpollen sculpturing of V. rupestris are similar to V. caspia (Figs. 2F, 4L, 5A), but the P/E ratioof V. rupestris is larger than V. caspia (Tab. 2).

Subsection Viola (Figs. 2G–I, 5B–I)

This subsection has three species in Iran, V. alba, V. sintenisii, and V. odorata. Whereasthe latter can be easily recognised using morphological characters, i.e. broad stipules andhigh number of leaf crenulae, the division between two first species is still problematic. Theshape in equatorial view of three species is prolate–spheroidal to subprolate (Figs. 2G–I).Pollen characteristics are also variable between the taxa, particularly, the exine orna-mentation shows that V. alba has perforate to subpsilate and perforate to granulate exine(Figs. 5B–C), V. sintenisii has perforate–granulate (Fig. 5H–I). Viola odorata has perforateto subpsilate and perforate to granulate exine (Figs. 5D–G).

Discussion

The exine sculpturing of the pollen grains, their size, shape and aperture numbers can beof taxonomic value in the studied taxa. The pollen size variability is a diagnostic valuebetween some of the taxa within the same section, for example, between V. arvensis and V.

kitaibeliana (sect. Melanium), V. modesta and V. occulta (sect. Melanium), V. cinerea and V.

stocksii (sect. Sclerosium).

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Fig. 5. SEM micrographs of pollen grains in Viola rupestris (A), V. alba (B–C), V. odorata (D–G), V.

sintenisii (H–I).

Fig. 4. SEM micrographs of pollen grains in Viola cinerea (A), V. stocksii (B–C), V. pachyrrhiza (D),V. spathulata (E–F), V. caspia (G–J), V. reichenbachiana (K), V. rupestris (L).



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Tab. 3. Characteristic features of the investigated pollen in species of Viola. M – mean value; SD – standard deviation

Species Sect. / Subsect.Colpate (mm)

Min (M ± SD) MaxExin thickness

(mm)Shape

(equatorial view)Shape

(polar view)

V. arvensis Melanium 52.5 (60.4 ± 4.8) 75.0 2.2 ± 0.2 Prolate spheroidala PentagonalV. kitaibeliana 35.0 (40.0 ± 2.8) 42.5 2.1 ± 0.2 Pyramidal TetragonalV. modesta 20.0 (24.5 ± 2.2) 27.5 2.3 ± 0.2 Prolate spheroidala TetragonalV. occulta 47.5 (53.9 ± 4.2) 62.5 2.5 ± 0.1 Prolate spheroidala TetragonalV. tricolor 42.5 (46.0 ± 1.7) 47.5 2.3 ± 0.2 Prolate spheroidala TetragonalV. somchetica Plagiostigma 20.0 (22.8 ± 1.6) 25.0 1.6 ± 0.2 Prolate spheroidala Circular-SubtriangularV. behboudiana Sclerosium 22.0 (23.3 + 1.4) 25.0 1.8 ± 0.1 Oblate spheroidal Circular-SubtriangularV. cinerae 16.5 (17.6 + 0.8) 18.7 1.2 ± 0.2 Prolate spheroidala Circular-SubtriangularV. stocksii 19.7 (19.8 + 0.1) 20.0 2.7 ± 0.2 Prolate spheroidala Circular-SubtriangularV. pachyrrhiza Spathulidium 20.0 (22.3 ± 1.6) 26.0 1.4 ± 0.1 Prolate spheroidala Circular-SubtriangularV. spathulata 20.0 (22.3 ± 1.6) 25.0 1.4 ± 0.1 Prolate spheroidala Circular-SubtriangularV. caspia 1 Viola.Rostratae 22.5 (27.9 ± 2.7) 32.5 1.8 ± 0.1 Prolate spheroidala Circular-SubtriangularV. caspia 2V. caspia 3

30.0 (31.5 ± 1.2) 33.830.0 (26.8 ± 2.7) 22.5

1.7 ± 0.12.0 ± 0.2

SpheroidalProlate spheroidala

Circular-SubtriangularCircular-Subtriangular

V. reichenbachiana 22.5 (28.0 ± 3.7) 35.0 2.4 ± 0.2 Oblate spheroidal Circular-SubtriangularV. rupestris 17.5 (21.6 ± 1.8) 25.0 2.2 ± 0.2 Prolate spheroidala Circular-SubtriangularV. alba subsp. alba 1 Viola.Viola 17.5 (21.8 ± 0.2) 25.0 2.0 ± 0.2 Prolate spheroidala Circular-SubtriangularV. alba subsp. alba 2 22.5 (24.6 ± 2.5) 30.0 2.2 ± 0.2 Prolate spheroidala Circular-SubtriangularV. alba subsp. alba 3V. odorata 1

20.0 (22.5 ± 1.8) 25.017.5 (20.8 ± 1.6) 22.5

2.0 ± 0.62.0 ± 0.2

Prolate spheroidalaProlate spheroidala


V. odorata 2 20.0 (21.2 ± 1.2) 20.0 2.2 ± 0.2 Subprolatea Circular-SubtriangularV. sintenisii 1V. sintenisii 2V. sintenisii 3

20.0 (21.0 ± 1.2) 22.522.5 (24.4 ± 2.1) 27.525.0 (27.5 ± 1.8) 30.0

1.8 ± 0.21.9 ± 0.21.5 ± 0.1

Prolate spheroidala

Prolate spheroidalaProlate spheroidala

Circular-Subtriangular




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Tab. 4. The number of aperture and pollen heteromorphism in the investigated species of Viola.

Species Sect. . Subsect.Number of apertures

(3 + 4 + 5)Percent of most abundant

pollen morph (%)Hetero-morphism

(<95 %)Predominant pollen

morph

V. arvensis Melanium 0 + 2 + 98 98 – 5 zonocolporate

V. kitaibeliana 0 + 97 + 3 97 – 4 zonocolporate

V. modesta 0 + 96 + 4 96 – 4 zonocolporate

V. occulta 0 + 85 + 15 85 + 4 zonocolporate

V. tricolor 0 + 97 + 3 97 – 4 zonocolporate

V. somchetica Plagiostigma 100 + 0 + 0 100 – 3 zonocolporate

V. behboudiana Sclerosium 92 + 8 + 0 92 + 3 zonocolporate

V. cinerae 99 + 0 + 0 100 – 3 zonocolporate

V. stocksii 98 + 2 + 0 98 – 3 zonocolporate

V. pachyrrhiza 98 + 2 + 0 98 – 3 zonocolporate

V. spathulata Spathulidium 98 + 2 + 0 98 – 3 zonocolporate

V. caspia 1 Viola/Rostratae 94 + 6 + 0 94 + 3 zonocolporate

V. caspia 2 97 + 3 + 0 97 – 3 zonocolporate

V. reichenbachiana 99 + 1 + 0 99 – 3 zonocolporate

V. rupestris 100 + 0 + 0 100 – 3 zonocolporate

V. alba subsp. alba 1 Viola/Viola 100 + 0 + 0 100 – 3 zonocolporate

V. alba subsp. alba 2 100 + 0 + 0 100 – 3 zonocolporate

V. alba subsp. alba 3V. odorata 1

100 + 0 + 0100 + 0 + 0

100100

––

3 zonocolporate3 zonocolporate

V. odorata 2 100 + 0 + 0 100 – 3 zonocolporate

V. sintenisii 1V. sintenisii 2V. sintenisii 3

100 + 0 + 0100 + 0 + 0100 + 0 + 0

100100100

–––

3 zonocolporate3 zonocolporate3 zonocolporate



19. oujak 2014 17:00:44



Pollen grains in polar view are variable within the sect. Melanium and the other sections(Tab. 2). A tetragonal to pentagonal shape in polar view is found in V. arvensis, V. kitaibe-

liana, V. modesta, V. occulta and V. tricolor (sect. Melanium), whilst circular-subtriangularis present in the other sections. The pollen shape of sect. Melanium was circular to sub-trian-gular, tetragonal or pentagonal in polar view and prolate to oblate, spheroidal or pyramidalin equatorial view according to the ratio of polar-equatorial axis (WALKER and DOYLE 1975).

According to the classification of pollen grains based on size (WALKER and DOYLE

1975), this section has large pollen grains (50–99 mm), while the other sections of Viola dis-tributed in Iran have medium sized pollen grains (25–49 mm).

Pollen grains with six apertures were reported for V. arvensis from Ukraine (GORB

1994). The figures given in ERDTMAN (1952) show that V. arvensis has 4-colpate with a few5-colpate grains appearing to be based on misidentification with V. tricolor since this type isfound in V. tricolor and not V. arvensis. PETTET (1964) noted that the two very variable andoften confused species, V. tricolor and V. arvensis, are readily separable on the basis of pol-len assemblage.

Earlier studies suggested that section Plagiostigma is not closely related to section Viola,as previously assumed (CLAUSEN 1929, MARCUSSEN et al. 2012). Our palynological resultsfor Iranian V. stocksii agree with findings for this species in Pakistan (PERVEEN and QAISER

2009).

Previously these species, i.e.. V. pachyrrhiza and V. spathulata were included in sectionPlagiostigma subsection Patellares (as section Nomimium grex Adnatae; BECKER 1918).However, this lineage takes a completely different phylogenetic position and will require asection of its own (MARCUSSEN et al. 2010). Yousefi et al. (2012) demonstrated that Viola

somchetica (section Plagiostigma) differs from V. spathulata (section Spathulidium) in thenumber of collenchyma layers at the corners of petiole cross sections (4 vs. 3) and the shapeof peduncle cross sections (quadrangular vs. circular).

Morphological and phylogenetic evidence supports two subsections, subsection Ros-

tratae, with aerial floriferous stems and explosive capsules, and subsection Viola, lackingaerial stems and with inexplosive capsules (BECKER 1925, MARCUSSEN and KARLSSON 2010).Anatomically, V. caspia can be separated from V. reichenbachiana by the presence of a pithregion in root cross-sections and by the number of vascular bundles in stem cross sections(YOUSEFI et al. 2012).

Viola behboudiana (section Sclerosium), V. caspia (section Viola) and V. occulta (sectionMelanium) showed structural polymorphism. The number of apertures in the two first oneswas 3–4, and 4–5 in the last one. All three species are high-polyploids. Viola behboudiana

and V. caspia are both octoploids (MARCUSSEN and BORGEN 2011), and this is probably thecase also for V. occulta (n = 10 versus n = 3, 4, 5, 7 in its close tetraploid relatives; YOCKTENG

et al. 2003). In the genus Viola, pollen heteromorphism is a direct result of polyploidy(NADOT et al. 2000).

Acknowledgements

We thank to Mr. A. Khodayari (University of Ardebil) for his assistance in electron mi-croscopy. This research was supported by a grant 989–27 from the research council of theUniversity of Guilan.

104 ACTA BOT. CROAT. 73 (1), 2014




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